Experimental and clinical observations have supported the concept that the hypothalamus plays a key role in the regulation of adenohypophysial corticotropic cells' secretory functions. Over 50 years ago it was demonstrated that factors present in the hypothalamus would increase the rate of ACTH secretion by the pituitary gland when incubated in vitro or maintained in an organ culture. However, a physiologic corticotropin releasing factor (CRF) was not characterized until ovine CRF (oCRF) was characterized in 1981. As disclosed in U.S. Pat. No. 4,415,558, the disclosure of which is incorporated herein by reference, oCRF was found to be a 41-residue amidated peptide. oCRF lowers blood pressure in mammals when injected peripherally and stimulates the secretion of ACTH and β-endorphin.
Rat CRF (rCRF) was later isolated, purified and characterized; it was found to be a homologous, amidated hentetracontapeptide as described in U.S. Pat. No. 4,489,163, the disclosure of which is also incorporated herein by reference. The amino acid sequence of human CRF was later determined to be the same as that of rCRF. rCRF and hCRF are used interchangeably to describe this peptide, and the designation r/hCRF is frequently used with respect to this peptide hormone. These peptide hormones are considered to form a part of a larger family of native CRF-like peptides and analogs which include the mammalian and fish CRFs, the urotensins and sauvagine, as discussed in Vale et al., “Characterization of the Hypothalamic Peptide: Corticotropin Releasing Factor”, Proceedings of the Naito International Symposium on Natural and Biological Activity, Tokyo, Japan, Nov. 5-7, 1985, and Lederis et al., “Neurohormones from Fish Tails, II: Actions of Urotensin I in Mammals and Fishes”, Recent Progress in Hormone Research, Vol. 41, Academic Press, Inc. (1985).
Although originally isolated and characterized on the basis of its role in this hypothalamo-pituitary- adrenal (HPA) axis, CRF has been found to be distributed broadly throughout the central nervous system as well as in extraneural tissues, such as the adrenal glands, placenta and testes, where it may also act as a paracrine regulator or a neurotransmitter. Moreover, the involvement of CRF in affective disorders, such as anxiety, depression, alcoholism and anorexia nervosa, and in modulating reproduction and immune responses suggests that changes in CRF expression may have important physiological and pathophysiological consequences. For example, perturbations in the regulatory loops comprising the HPA axis often produce chronically elevated levels of circulating glucocorticoids; such patients display the physical hallmarks of Cushing's syndrome, including truncal obesity, muscle-wasting, and reduced fertility.
In addition to its role in mediating activation of the HPA axis, CRF has also been shown to modulate autonomic and behavioral changes, some of which occur during the stress response. Many of these behavioral changes have been shown to occur independently of HPA activation in that they are not duplicated by dexamethasone treatment and are insensitive to hypophysectomy. In addition, direct infusion of CRF into the CNS mimics autonomic and behavioral responses to a variety of stressors. Because peripheral administration of CRF or a CRF antagonist fails to affect certain of these changes, it appears that CRF exhibits a direct brain action with respect to such functions, which include appetite suppression, increased arousal and learning ability. However, CRF antagonists given peripherally block endogenous CRF-mediated increases in ACTH secretion, and when such are delivered into the cerebral ventricles, stress-induced changes in autonomic activity and behavior can be mitigated.
As a result of the extensive anatomical distribution and multiple biological actions of CRF, this regulatory peptide is now believed to be involved in the regulation of numerous biological processes. CRF has also been implicated in the regulation of inflammatory responses. Although it has been observed that CRF plays a pro-inflammatory role in certain animal models, CRF appears to suppress inflammation in other models by reducing injury-induced increases in vascular permeability.
CRF analogs containing D-isomers of certain a-amino acids have been developed, such as those shown in U.S. Pat. No. 5,278,146. Synthetic r/hCRF and oCRF stimulate ACTH and β-endorphin-like activities (β-END-Li) in vitro and in vivo, and they substantially lower blood pressure when injected peripherally. Antagonists of these peptides and of sauvagine and urotensin are disclosed in U.S. Pat. No. 4,605,642, issued Aug. 12, 1986, the disclosure of which is incorporated herein by reference. Additional biopotent CRF antagonists have been developed, and are disclosed in U.S. Pat. Nos. 5,245,009; 5,493,006; 5,510,458; 5,663,292; 5,777,073; 5,874,227; and 6,323,312.
CRF antagonist peptides have been developed during the last 10-15 years which exhibit longer lasting and increased biological activity, in comparison to previously known CRF antagonists, and little or substantially no residual CRF agonist activity. Many of these exhibit high receptor affinity.
It has been shown that various of the members of the family of CRF-like peptides can be modified to create highly biopotent CRF antagonists that bind strongly to the known CRF receptors (CRF-R), including CRFR1 and CRFR2, without significantly activating such receptors and thus block the action of endogenous CRF at its receptors. They exhibit an affinity for CRFR1 and CRFR2 higher than that exhibited by oCRF. These modifications to create such bioactive CRF antagonists have included N-terminally shortening the native or other molecule so that it has a length of 30 to 33 residues, e.g. r/hCRF(9-41), r/hCRF(10-41), r/hCRF(11-41) and r/hCRF(12-41), and incorporating a cyclizing bond, preferably a lactam, which joins the side chains of the residues that are located in the positions of the 8th and 11th residues from the C-terminal residue, e.g. (cyclo 30-33)[Glu30, Lys33]r/hCRF(12-41). It was found that such a cyclizing modification often very substantially increased the biopotency of the comparable linear peptide. It was also found that the combination of this cyclizing bond plus the acylation of the N-terminus created a molecule of long-acting duration and that such an effect may be greatest in a peptide of 33 residues in length, e.g. (cyclo 30-33)[Ac-Asp9, Glu30, Lys33]-r/hCRF(9-41). The family of CRF-like peptides is generally considered to encompass those peptides which bind to the CRF receptors and have at least about 45% amino acid structural homology with ovine CRF, the first mammalian CRF isolated and characterized. The CRF-like family includes, but is not limited to, the following known peptides: ovine CRF, rat/human CRF, porcine CRF, bovine CRF, fish CRFs, carp urotensin, sucker urotensin, maggy sole urotensin, flounder urotensin, sauvagine, the urocortins 1, 2 and 3, and stresscopins.
Efforts for improving CRF antagonists have generally concentrated on increasing affinity for one or the other known receptors (CRFR1 and CRFR2) or for both receptors (CRFR1/2) with just one antagonist. Scant effort has been expended at optimizing the physical/chemical properties towards obtaining clinically safe, potent, stable, inexpensive-to-make analogs, i.e., improving “drugability” of such analogs. The search for CRF antagonists having ever-improved drugability continues.